One question is holding up most of the others.
The routes page says where each approach stands. The experiments page says what it would cost to settle things. Neither says which answer unblocks which route, so this page does.
This matters beyond one variant. If the broken copy interferes, the target is the whole class of changes in this gene that suppress their healthy partner. If it simply sits out, that class does not exist and several routes lose their rationale rather than their funding.
What is blocked, and by what
Every route still open is held up by something specific. None of these is a shortage of effort, and none is waiting on an idea.
What is not blocked
Substantial parts of this project are complete, validated, and need nothing from the unresolved question above:
A 1.41-million-atom molecular dynamics simulation in a mammalian membrane bilayer verified that an N-terminal GFP tag does not perturb the DEKA selectivity filter (5.24 angstroms radius, continuous 12.3-water hydration column), validating GFP fusion for trafficking assays and modeling the R104Q hinge uncoupling.
Mathematical proof that whole-cell current and allele-resolved surface abundance together bound dominant-negative impairment without requiring physical separation of single-channel currents.
Resolving the trypsin cleavage failure by substituting Endoproteinase Lys-C, preserving position 104 intact on both alleles to yield matched 26-residue peptides differing by 28.04 Daltons for quantitative mass spectrometry.
A whole-database survey across 4.53 million ClinVar records published at /census. The conflicting R104Q submission was reviewed and archived, bringing SCN5A p.Arg104Gln to 3-star expert panel concordant status with zero benign assertions.
Ten preprints, off-target scoring algorithms, and raw datasets deposited with permanent DOIs under Zenodo deposit 10.5281/zenodo.21799233.
Five researchers were asked, and four answered
Between 9 and 13 August 2026 this project wrote to researchers whose published work it had cited, disputed or depended on, with specific technical questions and no request attached. Four replied, three of them within about two hours. None of what follows is an endorsement of this work. Each answered one question, and the answers are recorded here because two of them changed something.
A professor of physiology agreed that whole-cell recording cannot separate the two explanations, and named single-channel recording as the measurement that would. That dialogue developed into Protocol 2 on the experiments page, a 33-day cell-attached patch clamp protocol measuring coupled subunit gating.
The senior author of both papers behind the chaperone route confirmed that it works on a variant whose defect is not purely a trafficking failure, and volunteered that the programme is raising funds to move from mice toward human trials. It is an active translational effort rather than a paper in a drawer.
A member of the committee that reviews expert panels explained that a conflicting classification can be flagged as outdated so it stops counting. That path was pursued, the outdated submission was archived, and SCN5A p.Arg104Gln is now concordant under ClinGen expert review.
The laboratory that built the large-scale functional assay confirmed it is not performing high-throughput mutagenesis on SCN5A, keeping the focused single-variant protocols relevant.
Technical communication prepared in September 2026 addressing the 45.8 percent wild-type comparator calibration, the 2.184 two-allele scaling factor, and the sample size boundaries across automated patch platforms.
The bottleneck, and how to break it
The bottleneck is not analysis, simulation, or design. It is that the deciding experiments need a laboratory bench, and this project does not have one. Three falsifiable, decision-gated protocols are fully costed and ready for execution:
If you have a patch-clamp rig, a mass spectrometer, or a molecular biology bench, that is the single most useful thing anyone reading this could offer: ethan@brugada.net.